The Extracellular Matrix Environment of Clear Cell Renal Cell Carcinoma Determines Cancer Associated Fibroblast Growth

Abstract

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer and is often caused by mutations in the oxygen-sensing machinery of kidney epithelial cells. Due to its pseudo-hypoxic state, ccRCC recruits extensive vasculature and other stromal components. Conventional cell culture methods provide poor representation of stromal cell types in primary cultures of ccRCC, and we hypothesized that mimicking the extracellular environment of the tumor would promote growth of both tumor and stromal cells. We employed proteomics to identify the components of ccRCC extracellular matrix (ECM) and found that in contrast to healthy kidney cortex, laminin, collagen IV, and entactin/nidogen are minor contributors. Instead, the ccRCC ECM is composed largely of collagen VI, fibronectin, and tenascin C. Analysis of single cell expression data indicates that cancer-associated fibroblasts are a major source of tumor ECM production. Tumor cells as well as stromal cells bind efficiently to a nine-component ECM blend characteristic of ccRCC. Primary patient-derived tumor cells bind the nine-component blend efficiently, allowing to us to establish mixed primary cultures of tumor cells and stromal cells. These miniature patient-specific replicas are conducive to microscopy and can be used to analyze interactions between cells in a model tumor microenvironment.

Cancer-Associated Fibroblasts: Mechanisms of Tumor Progression and Novel Therapeutic Targets

“…This appears to be due, in large part, to the activation of pathways that regulate tumor cell viability and aggressive behavior. Indeed, stromal nicotinamide N-methyltransferase (NNMT) is necessary and sufficient for CAF function (i.e., expression of CAF biomarkers, cytokine secretion, deposition of an oncogenic ECM [ 113 ]) in high-grade serous carcinoma [ 114 ]. Elevated NNMT expression defined, in part, the metastatic stromal proteome and supports ovarian cancer proliferation, migration and distal spread.…”

The application of 3D bioprinting in urological diseases

“…The proteomics are employed to identify the ECM compositions of urologic cancers. Clear cell renal cell carcinoma (ccRCC) is found that the collagen type VI, fibronectin, and tenascin C are significantly upregulated compared with healthy kidney cortex [ 165 ]. CRPC has upregulation of collagen type I, fibronectin, vitronectin, and laminin compared with normal prostate tissues [ 166 ].…”

Targets for Renal Carcinoma Growth Control Identified by Screening FOXD1 Cell Proliferation Pathways

“…Three-dimensional fibrin cultures of 786-O, 786-O FOXD1null , and primary RCC cell lines were created following our previously reported protocol [ 9 ]. In summary, 3D cultures were generated by combining fibrinogen (MilliporeSigma, Burlington, MA, USA cat#F8630) at a 1:1 ratio with cells suspended in a culture media, with or without the addition of ECM proteins.…”

“…All tissue specimens and associated medical information received were de-identified using an honest broker system. Samples were processed as previously described [ 9 ]. Fresh tumor tissue samples were diced and weighed.…”

“…We identified candidates with known functions related to the FOXD1 null phenotype, and compounds that modulate their function were evaluated for their potential to replicate the cell cycle phenotype caused by the loss of FOXD1 . A panel of patient-derived 3D tumor replicas was developed using a method recently described by our group to validate the effects of candidate compounds in clinically relevant models [ 9 ]. Our study uncovers FOXM1, PME1, and TMEM167A as potential targets for pharmaceutical intervention to slow ccRCC tumor cell growth and provides a strategy for patient-specific compound testing.…”